In this paper, the interaction behavior of p-sulfonated calix[n]arene (SCnA) with tramadol hydrochloride was investigated by fluorescence spectra, 1 H NMR spectra, and theoretical calculations in aqueous solution. At the optimized conditions, the fluorescence intensity of tramadol hydrochloride showed positive correlation to the concentration of SCnA, which led to a validated, simple, and sensitive fluorescence quenching method for the determination of tramadol hydrochloride was established for the first time. Moreover, the interaction based on SCnA superstructure provided has promising potential for therapeutic monitoring and pharmacokinetics and clinical application